This invention relates to the enzyme Peptide-N.sup.4 -(N acetyl-.beta.-N-glucosaminyl) asparagine aminidase (hereinafter, PNGase).
Tarentino et al. 257 J. Biol. Chem. 10776, 1982 describe a peptide: N-glycosidase isolated from almond emulsion (hereinafter PNGase A). They state that this enzyme has potential for use in structural analysis of glycopeptides.
Plummer et al. 259 J. Biol. Chem. 10700, 1984 state that Endo-.beta.-N-acetylglucosaminidase F (hereinafter, Endo F) preparations from Flavobacterium meningosepticum also contain a peptide N-glycosidase activity (PNGase F). This PNGase activity cleaves the .beta.-aspartylglycosylamine linkage of N linked carbohydrate chains from glycoproteins, releasing a full length, or intact, oligosaccharide. All the Endo F preparations were mixtures of Endo F and PNGase F activities. Partial separation of the two enzyme activities was achieved by differential ammonium sulfate precipitation, and column chromatography.
Tarentino et al. 24 Biochem. 4665, 1985 describe separation of Endo F and PNGase F by ammonium sulfate precipitation and gel filtration on TSK HW-55(S). The PNGase F activity obtained is stated to be nearly free of Endo F (containing 0.4% Endo F activity). The authors state that deglycosylation of native proteins by PNGase F can provide a useful approach for investigating structure function studies of biologically active glycoproteins.
Hirani et al. 162 Analytical Biochemistry 485, 1987 describe use of PNGase F to release asparagine-linked oligosaccharides for structural analysis. They state that the structure of the released oligosaccharides can be analyzed by HPLC.
Plummer et al. 163 Eur. J. Biochem. 167, 1987 describe PNGase activities in lentil, split pea, pinto bean, lima bean, barley, and raw wheat germ.
Tarentino et al., 138 Methods in Enzymology 770, 1987 describe a standard protocol for isolation of PNGase F activity, and an assay for that enzyme activity.
Referring to FIG. 1, the different activities of PNGase and Endoglycosidase F (Endo F) on glycoproteins and glycopeptides are shown. A PNGase cleaves the bond between an N acetylglucosamine residue and an asparagine residue, while Endo F cleaves the bond between two adjacent N acetylglucosamine residues. Thus, PNGase provides a full length carbohydrate chain, while Endo F provides a shortened chain.